1. Field of the Invention
The present invention relates to analytical methods and systems for measuring the rate of biomolecular reactions. More particularly, the invention has to do with the use of electrochemiluminescence ("ECL") to monitor in real time the progress of a biomolecular reaction. The method can be used to monitor the progress of affinity binding reactions and, as such, can be used in antibody-antigen binding rate measurements, among others. The method also can be used for the diagnostic determination of an enzyme activity or concentration and for other rate measurements as will be apparent to those skilled in the art.
2. Description of Related Art
There are various known methods for measuring the progress of biomolecular reactions and the present invention provides a new method of monitoring the rates of such reactions. The progress of enzymatic reactions, for example, has been monitored by spectrophotometry and fluorescence. These methods and others are used in modern laboratories and have been used by applicants to obtain reference data for the development of the new analytical technique of the present invention.
Antigen-antibody reaction rates can be measured using a technology called real-time biospecific interaction analysis which uses surface plasmon resonance to detect biomolecular interactions. The method was reported to be a valuable supplement to conventional methods of investigation in an article entitled "Label-Free Biosensor Technology Visualizes Biomolecular Interactions in Real Time", Biosensors & Bioelectronics, Vol. 8, No. 2, Products and Innovations, pp. xi-xiv. The use of surface plasmon resonance is also discussed by Sjolander, S. and Urbaniczky, C. in "Integrated Fluid Handling System for Biomolecular Interaction Analysis", Analytical Chemistry 1991, Vol. 63, pp. 2338-2345. According to the method, the kinetics for biomolecular interactions between an antigen and an antibody can be followed directly without labeling. The method is useful for detecting, in situ, low concentrations of biochemically active molecules having high molecular weight.
A general procedure for the determination of the dissociation constant (K.sub.D) of antigen-antibody equilibria in solution is reported by Friguet, B., et al., in "Measurements of the True Affinity Constant in Solution of Antigen-Antibody Complexes by Enzyme-Linked Immunosorbent Assay", Journal of Immunological Methods 1985, Vol. 77, pp. 305-319. The method employs a classical indirect ELISA and is reported to permit the detection of very small concentrations of antibody and the determination of K.sub.D values as small as 10.sup.-9 M.
The method and system of the present invention employ electrochemiluminescence which has been used heretofore in analytical methods for the qualitative and quantitative analysis of chemical moieties. In U.S. Pat. No. 5,310,687, for example, a chemical moiety is disclosed which comprises a chemical, biochemical or biological substance attached to one or more electrochemiluminescent organometallic compounds. Methods are disclosed for detecting low concentrations of the chemical moiety using chemiluminescent, electrochemiluminescent and photo-luminescent means. Compounds are disclosed which are useful for labeling substances of interest with ruthenium-containing and osmium-containing labels or other electrochemiluminescent labels. The labeled substances are useful in methods for detecting and quantifying analytes of interest in binding assays and competitive binding assays.
We have now discovered a method and system of employing electrochemiluminescence to monitor the progress of biomolecular reactions and the method can be employed in diagnostic kits for clinical use, research laboratories, and the like. The method employs commercially available equipment and provides a highly accurate means for diagnostic determination of an enzyme activity or concentration. The method also provides a means to measure antibody-antigen binding rates and it is useful for screening for high binding rate antibodies. In one embodiment, a method has been derived for measuring the rates of antibody binding to carcinoma embryonic antigen. In another embodiment, a method has been derived to determine lactate dehydrogenase for clinical applications.